Retinoblastoma is a malignancy of the developing retina caused by mutation of both alleles of a single gene (RB1). RB1 is the prototypical tumor suppressor gene, a gene whose inactivation leads to tumorigenesis. Not only does mutation of RB1 cause retinoblastoma, but its mutation has been detected in a variety of common human neoplasia. These include carcinoma of the breast, lung, prostate, bladder, and cervix. For these reasons it has become the focus of efforts to understand the molecular mechanisms that regulate a cell's transition to malignancy. RB1 function, if it could be appropriately manipulated, might serve as a means to treat relevant cancers. A prerequisite for this approach is understanding how RB1 regulated. The RB1 gene product (110RB) is a nuclear phosphoprotein which negatively regulates progression through G1 phase of the cell division cycle. Phosphorylation of 110RB occurs in synchrony with the cell cycle, suggesting that 110RB function may be regulated by cell cycle-dependent phosphorylation. The cyclin-dependent kinases (cdks) are good candidates for kinases that regulate 110RB due to the characteristic oscillation of their activity during the cell cycle. Previously, we have shown that microinjection of unphosphorylated 110RB, or a truncated form (p56RB), in early G1 cells will block entry into S phase. Using this assay, we propose to directly assess the consequences of phosphorylaiton by G1 and S phase cyclin/cdk complexes on the ability of RB1 protein to negatively regulate S phase entry. The hypothesis addressed in this proposal is that phosphorylation by specific cyclin dependent kinases inhibits 110RB function. The specific aims of this proposal are to; 1) produce preparations of purified 110RB that have been phosphorylated in vitro by cyclin-dependent kinases; 2) test the ability of these differentially phosphorylated 110RB preparations to inhibit cell cycle progression; 3) determine the particular phosphorylation target sites important for regulation by cdks; 4) characterize the functional consequences of mutation at these sites; 5) test the ability of these cdks to regulate the cell cycle arrest activity of RB1-like protein p107. Preliminary results indicate that in vitro phosphorylation of 110RB by cyclin D1/cdk4 (D1/k4), cyclin E/cdk2 (E/k2), and cyclin A/cdk2 (A/k2) causes the characteristic mobility shift upon resolution by SDS-PAGE, denoting hyperphosphorylation. Hyperphosphorylation of 110RB by D1/k4 inhibits its ability to arrest the cell cycle. However, RB1 protein hyperphosphorylated by A/k2 or E/k2 retains its ability to block entry into S phase. Tryptic phosphopeptide mapping of 110RB treated by these kinases revealed a complex pattern of phosphorylation. While the pattern produced by phosphorylation with D1/k4 partially overlaps that produced with E/k2 and A/k2, D1/k4 phosphorylates sites which lead to four tryptic phosphopeptides only weakly phosphorylated by E/k2 and A/k2. The long term objectives of this proposal are to understand how phosphorylation regulates RB1 function, which kinases are capable of regulating RB1, and how this relates to regulation of other RB1-like proteins.